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Metagenomics to Study Clostridium difficile Resistance
Public Health and ''C. difficile'' Infection with C. difficile is a significant cause of hospital morbidity and mortality, causes extensive diarrhea and dehydration and is associated with antibiotic resistance. In many people, C. difficile lives in harmony with other "healthy" bacterial populations. The complex populations of other bacterial species keep C. difficile populations in check. Systemic antibiotics indiscriminately kill off our gut bacteria, which then allows C. difficile to become more abundant. This is a common route to C. difficile ''infection. A successful treatment to restore microbiota complexity involves a fecal microbiota transplant from a healthy individual. The fecal transplant method can even treat recurrent ''C. difficile ''infections. Little is known about which bacterial populations in fecal transplants are responsible for the recovery of individuals infected with ''C. difficile''Buffie, Charle, et al., 2014, Precision microbiome reconstitution restores bile acid mediated resistance to ''Clostridium difficile, Nature, ''http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13828.html ''. Study Rationale In recognition of the burgeoning public health crisis associated with C. difficile infection, the authors of this study set out to use metagenomic techniques to study mammalian gut microbiota changes, and then assess which microbiome-encoded genes were associated with resistance to C. difficile. ''Prior to this study, the underlying bio-synthetic microbiome-encoded genes and gene clusters that were required for an individual's resistance to infection were undefined. This information is necessary to understand effective therapies that treat imbalanced microbiota or target specific bacterial populations within the microbiota. This study: ''1: Studied the impact of several commonly used antibiotics on microbiota 2: Identified the bacterial populations important for resistance 3: Verified the importance of these populations, and confirmed the specific genes associated with their ability to resist C. difficile infection. Impact of Common Antibiotics on Microbiota '' Three separate mice groups were used. Each colony recieved one of three antibiotics: clindamycin, ampicillin or enrofloxacin. Interesting methodological note: clindamycin was administered via injection, while ampicillin and enrofloxacin were administered through drinking water. Anyway, following the ending of an antibiotic regime, which would simualte a typical human regime, a mouse from each of the groups was infected with C. difficile ''at each of the times (days 1, 6, 10, 14, and 21). The feces were examined using metagenomic techniques looking at 16S amplicon sequences. This part of the study confirmed prior findings from this research group. These findings are: A clindamycin regimen result in long period of susceptibility to ''C. difficile infection (first row A and B). An ampicillin regimen had a brief (around 6 day period of) susceptibility to C. difficile infection (second row C and D). Enrofloxacin regimen did not cause an increase the susceptibility of infection with C. difficile (''third row E and F). The data is not shown, but the expression of ''C. difficile toxin was significantly correlated with the amount of intestinal ''C. difficile. ''Based on figures B, D and F, it can be somewhat observed that the antibiotics (clindamycin, ampicillin, enrofloxacin) did not cause a significant alteration of bacterial density, but rather of the amount of different bacterial species. ''Metagenomic'' Identification of C. difficile Resistance Bacteria '' From the same experiment discussed above, the microbiota were identified prior to administration of antibiotics, and then afterwards. The microbiota of mice that were infected with C. difficile ''(called Susceptible) were compared to the microbiota of mice that resisted infection (called Resistant). An interesting finding was that while resistance was correlated with an increased diversity of microbiota, a highly diverse microbiota was not necessarily ''sufficient to resist infection. Note the circled mice that were both resistance and susceptible to C. difficile ''infection, and have a low biodiversity. A common held belief is that a highly diverse microbiota population increases resistance. These data suggested a more complex story than just a diverse microbiota alone for resistance. Indeed, using the 16S identification of different bacterial species, it was found that some populations had a strong negative interaction against ''C. difficile ''infection, while other bacteria populations had a highly positive interaction. The negative bacteria that were selected as the best candidates to study and that had a potential to imbue resistance to ''C. difficile ''infection were ''C. scindens, ''Barnesiella intestihominis,'' Pseudoflavonifractor capillosus and Blautia hansenii. Interestingly Streptococcus thermophilus and Enterococcus avium were found to have the highest positive interaction with C. difficile ''infection. 'Holistic, Metagenomic Verification of Resistance Populations' As in the previous experiment, three separate groups of mice were administered antibiotics. This time, shortly after the antibiotic regime ended, the mice received either a suspension of four-bacteria cocktail containing equal parts, ''C. scindens, Barnesiella intestihominis, Pseudoflavonifractor capillosus, and Blautia hansenii, ''just ''C. scindens, or a control of PBS solution. For the curious out there, PBS stands for Phosphate Buffered Saline (a great buffer). These three groups were given their treatment for 2 consecutive days before exposed to C. difficile. ''Then, the quantity of ''C. difficile bacteria were quantified from the mice feces. As shown, the results validated the initial metagenomic findings that these particular bacterial species were important for resistance to C. difficile ''infection (Figure A). ''C. difficile ''resistance in the two active transfer groups (4-bacteria cocktail and ''C. scindens ''alone) versus infection in the control was was confirmed by testing for ''C. difficile molecular toxin (Figure B). Only the 4-bacteria cocktail seemed to negate weight loss, although all groups were restored to initial weight by 20 days(Figure C). Perhaps most compelling, the overall survival was determined less than 10 days after infection, and the 4-bacteria cocktailed group had 100% survival, the C. scindens group had the second best survival, and the control group the worst(Figure D). The C. scindens ''was found to have the most strongest negative interaction with ''C. difficile.'' Blautia hansenii was found to have the weakest negative interaction with ''C. difficile (''Figure E''). ''There was not a significant difference in biodiversity before antibiotic treatment and after antibiotic treatment in any of the three groups, ''including in the PBS Control ''(Figure F). '''Microbiota Resistance Gene Confirmation' Based on the previous experiments, it was found that secondary bile acids were more abundant in resistant mice. Secondary bile acids are secreted by bacterial in the colon. Relative abundance of secondary bile acid species is shown to the left in Figure A. This prompted the research group to investigate the etiology of secondary bile acids further using more metagenomic techniques. Using shot-gun sequencing techniques, the genes involved in secondary bile acid synthesis were probed in resistant and susceptible individuals. It was found that a key step to get secondary bile acids involved 7-Dehydroxylation, which is the responsibility of the bai operon. The '' susceptibility of ''C. difficile ''infection is shown on the X-Axis by Spearman correlations. In this case, a negative correlation means that expression of these genes is associated with resistance to ''C. difficile ''infection (Figure C). The key secondary bile acid product, which is formed as a result of the bai operon is deoxycholate (DCA). In Figure E, it can be observed that having the bai operon (+) or not (-) determined the quantity of DCA. This graph also shows the positive correlation between the bai operon, ''C. scindens, again demonstrating the link between C. scindens ''and resistance to ''C. difficile ''infection. The key confirmation of these findings can be found in the supplemental data. A cell culture of ''C. difficile ''was treated with DCA. ''C. difficile ''was inhibited in a dosage-dependent manner (Lower Figure A). The vehicle is the control. A dose-dependent response in a biological system is strong evidence for causality. This is the power of metagenomicsBuffie, Charle, et al., 2014, Precision microbiome reconstitution restores bile acid mediated resistance to ''Clostridium difficile, Nature, ''http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13828.html. ''See the full manuscript published in Nature: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13828.html References